Purification method and apparatus

ABSTRACT

PCT No. PCT/GB94/01484 Sec. 371 Date Jan. 4, 1996 Sec. 102(e) Date Jan. 4, 1996 PCT Filed Jul. 8, 1994 PCT Pub. No. WO95/02049 PCT Pub. Date Jan. 19, 1995A method is provided for purifying from cells a target compound such as nucleic acid. The method comprises the following steps: 1) lysing a cell suspension to form a cell lysate containing the target compound; 2) applying the cell lysate to a filter to remove unwanted cells and cell debris; 3) contacting the filtered lysate with a solid phase matrix under conditions to bind the nucleic acid to the matrix; 4) separating the resultant filtered lysate from the matrix; and 5) eluting the target compound from the matrix. Apparatus is also provided. Complex purification procedures such as centrifugation are avoided.

The present invention relates to a method and apparatus for purifyingtarget compounds such as nucleic acids from cells.

Conventional procedures for the purification of protein or nucleic acid,such as DNA, require lysis of the source cells followed by variousfractionation steps involving centrifugation. Where DNA manipulation isto be carried out, small scale DNA preparations are required routinely,often in large quantities for the purpose of screening the DNA from thesource cells. These processes are time consuming and labour intensive.

It has been proposed to avoid centrifugation in the extraction andpurification of DNA by a relatively complicated series of steps. EP0376080 discloses in general terms the use of precipitants toprecipitate DNA from accompanying impurities. Ultrafiltration is thenused as a means to isolate the DNA. However, no worked example of theproposed process is described and no indication of its feasability isindicated. Ultrafiltration forms the basis of other DNA purificationmethods as described in WO 87/07645 and EP 0517515.

Cation exchange resins have also been proposed as a means for separatingrelatively uncontaminated nucleic acids from impurities. EP 0281390discloses the use of polycationic solid supports particularly toseparate hybridized from unhybridized nucleic acids. EP 0366438discloses the separation of nucleic acid from protein by binding theprotein to a cation exchange resin.

A partially automated apparatus for carrying out biochemical reactionsis disclosed in WO 92/02303 in which mountings for manipulatingmicrotiter plates are proposed.

The present invention aims to provide an improved method for thepurification from cells of target compounds such as nucleic acids, whichmethod avoids the use of centrifugation, ultrafiltration or othercomplex procedures.

The target compound may comprise nucleic acid, protein or other desiredcompounds and is produced by the cell and expressed either internally orexternally. Apart from nucleic acids, the method is particularlyattractive for purifying recombinant proteins and antibodies.

In one aspect, the present invention provides a method for purifyingnucleic acid from cells. The method comprises the following steps:

(1) lysing a cell suspension to form a cell lysate containing nucleicacid;

(2) applying the cell lysate to a filter to remove unwanted cells andcell debris;

(3) contacting the filtered lysate with a solid phase matrix underconditions to bind the nucleic acid to the matrix;

(4) separating the resultant filtered lysate from the matrix; and

(5) eluting the nucleic acid from the matrix.

Because the method of the present invention is relatively simple,starting with the cell suspension it can be applied as a continuousmethod (i.e. without interruption from batch of cell suspension todesired product), for example in automated apparatus.

In a further aspect, the present invention provides a continuous methodfor purifying a target compound from cells. The method comprises thefollowing steps:

(1) obtaining a cell suspension containing the target compound;

(2) applying the suspension to a filter to remove unwanted cells andcell debris;

(3) contacting the filtrate with a solid phase matrix under conditionsto bind the target compound to the matrix;

(4) separating the resultant filtrate from the matrix; and

(5) eluting the target compound from the matrix.

Preferably, the cell suspension is lysed in step (1) to form a celllysate containing the target compound.

Preferably, the method further comprises the step of washing the matrixbinding the target compound so as to remove contaminants before elutingthe target compound from the matrix.

Any cell producing the target compound may be used in the method. Forthe purpose of this specification, the term "cell" is intended toencompass bacterial cells, cells from higher organisms, phage particlesand other cell types or organelles which contain the target compound andmay require some form of lysis step to release it. In the case ofbacteria, nucleic acid may come from the bacterial nucleus or fromcellular inclusions such as plasmids. Indeed, the method is especiallyuseful for the preparation of plasmid DNA. Phage-infected bacteria mayalso be used in the preparation of phage DNA, such as Ml3 DNA. Cellsfrom higher organisms include blood cells. Where genomic nucleic acid isto be prepared, nucleated blood cells form the cell suspension forlysis.

As a typical first step in the method, a cell suspension is formed byapplying a cell culture to the filter so as to separate the cells fromthe culture medium. The liquid medium passes through the filter and isdiscarded. The cells on the filter are resuspended in a suitableresuspension buffer ready for lysis. However, in the case of phage,depending on the type of phage, it may be necessary to introduce anextra filtration step so as to clear phage cells (particles) from thecell debris. For example, where lambda phage infects bacteria, someinfected bacteria lyse and some do not. In this case, it is necessary touse the extra filtration step as a pretreatment. In the pretreatment,the initial mixture of lysed bacterial cells, unlysed bacterial cellsand unlysed phage cells (particles) is typically filtered through apretreatment filter to remove unlysed bacterial cells and cell debris.The resultant phage cells are then kept in suspension ready for lysis,along the lines described below. Preferably, the suspension of phagecells is concentrated before lysis. Concentration may be effected bytreatment with salt and a hydrophobic agent such as polyethylene glycol(PEG). In contrast, where phage Ml3 infects bacteria, no lysis of thebacterial cells occurs and so no extra filtration step is required.

The step of lysing the cell suspension typically requires the additionof a further aliquot of lysis buffer. After lysis, it is sometimespreferable to add a neutralization solution to the lysed cellsuspension. Typically, the buffers for resuspension and lysis and theneutralization solution are all well-known in this field and can varyaccording to the cell type and target compound to be purified. Forexample, where nucleic acid is to be purified the resuspension buffertypically contains a chelating agent to remove metal ions from themedium and may a have pH in the range 7 to 8.5, advantageously in therange 7 to 8. For plasmid purification the lysis buffer is typicallyalkaline and includes a surfactant such as sodium dodecylsulphate (SDS).The neutralization solution is intended to bring the pH back into auseful range and can flocculate unwanted protein and is typically ahighly concentrated salt solution such as potassium acetate at around2.5 M. For nucleic acid purification from blood cells, the lysis bufferincludes NonIdet P-40 as a typical surfactant and the pH is preferablyraised to around pH 8.3.

The nucleic acid may be DNA or RNA. Where DNA is to be purified, theresuspension buffer may contain RNase to cleave unwanted RNA. Where RNAis to be purified the resuspension buffer may contain DNase to cleaveunwanted DNA. Optionally, for nucleic acid purification the resuspensionbuffer may also contain proteases, for example proteinase K.

At each stage of adding the resuspension buffer, lysis buffer, andneutralization solution, it is preferable to mix the solutions welltogether.

After lysis, the cell lysate is applied to the filter to remove unwantedcells and cell debris thereby giving rise to a crude preparation of thetarget compound and other soluble contaminants. Any filter commonlyavailable may be used in this step provided that the filter can toleratethe reagents being used and provided that the unwanted cells and celldebris are retained by the filter. For example, the filter may be madeof cellulose acetate, PTFE, or any other similar material. Preferably,the pore size of the filter is no greater than 50 microns. Too large apore size lets through unwanted matter. If the pore size is reducedbelow 0.2 microns the flow rate through the filter is disadvantageouslylow. In practice, the pore size is chosen in accordance with the celldensity and nominal cell size.

The solid phase matrix used in the present method must bind the targetcompound under the operating conditions used. The operating conditionsmust therefore be tailored in accordance with the type of matrix and thetarget compound. For nucleic acid, the matrix is typically glass- orresin-based and can bind the nucleic acid by ionic interaction, affinityinteraction or hydrophobic interaction. Advantageously an ion exchangematerial is used, preferably an ion exchange resin commerciallyavailable for this purpose. Cation exchangers are preferred ionexchangers on the basis that, at typical working pHs, the target nucleicacid is negatively charged. Possible cation exchangers include anysuitable proprietary resins (such as the "Magic" or "Wizard" range fromPromega Corp. or Sephacryl from Pharmacia AB) or borosilicate glassexchangers or diatomaceous earth. As a further option, a poly-adenylenecolumn may be used.

In each case, the conditions of pH and salt concentration will affectboth the ability of the target compound and the unwanted contaminants tobind to the matrix. It is therefore advantageous to include the step ofwashing the matrix prior to elution so as to remove unwantedcontaminants which bind less strongly to the matrix than the targetcompound. A typical wash buffer for nucleic acid is alcoholic Tris-HClat around pH 7.5 with 200 mM sodium chloride and 5mM EDTA.

The contacting of the filtrate with the solid phase matrix may occursimply by passing the filtrate through a volume of the matrix, perhapsin the form of a column, for example. Alternatively, a suspension of thematrix may be added to the filtrate. Separation of the thus-treatedresultant filtrate from the matrix may be effected simply by retainingthe matrix and discarding the unwanted liquid phase.

Elution of the target compound from the matrix will depend upon thenature of the interaction between the compound and the matrix. Whereionic interactions predominate, it is usual to elute the nucleic acid inan elution buffer having lower salt concentration. Higher pH or highersalt is generally required for proteins. Alternatively, the elutionbuffer may contain an affinant designed specifically to elute the targetnucleic acid or protein.

It is generally advantageous to keep the target compound in solutionwhen not bound to the solid phase matrix. The method is thereforepreferably carried out substantially in the absence of any precipitationof the target compound.

In contrast to the general methodologies described above, where thetarget compound to be purified is Ml3 DNA a modified method ispreferred. In this case, the source "cells" comprise Ml3 phageparticles. Bacterial cells infected with the Ml3 phage are filtered toremove unwanted bacterial cells and bacterial cell debris so that thefiltrate contains the Ml3 phage particles. The phage particles areprecipitated with any suitable precipitant, such as glacial acetate acidand subsequently lysed by contact with a suitable chaotropic lysissolution such as concentrated NaClO₄. The lysed Ml3 phage particles nowpresent in the filtrate are contacted with a solid phase matrix suitablefor binding the phage DNA. The matrix may be contacted as describedabove or, in a convenient embodiment, may be present in the form of afilter to which the lysed phage particles bind such as a borosilicateglass filter. The matrix is advantageously washed with a suitablewashing solution, such as an ethanol solution, and the DNA eluted asdescribed above.

In a further aspect of the present invention apparatus is provided forpurifying a target compound from a cell suspension. The target compoundmay be a target macromolecule such as protein or nucleic acid,preferably a nucleic acid. The apparatus comprises

a first chamber to receive the cell suspension;

a filter downstream of the first chamber for retaining unlysed cells andcell debris; and

a second chamber to receive filtrate downstream of the filter andcommunicating with means for delivering solutions thereto;

wherein means are provided in or downstream of the second chamber forretaining a solid phase matrix to bind the target compound and an outletfor delivering the purified target compound is provided downstream ofthe means for retaining the solid phase matrix.

Preferably, the first chamber communicates with further means fordelivering solutions thereto.

The apparatus can be incorporated into an automatic system comprisingone or more apparatus arrangements as described, together with a centralcontrol means for controlling the apparatus. The apparatus isparticularly suitable for operating the method described above and can,in automated form, permit the simultaneous operation of a plurality ofseparate purifications in a routine manner.

Each means for delivering solutions to the respective chambers maycomprise any suitable device for liquid delivery. For example, a syringemay be provided for the delivery of each buffer solution. Alternatively,a plurality of reservoirs containing the appropriate reagents may beconnected by valves to each chamber, for example through a common port.In the automatic version of the apparatus the control means would belinked to the valves to ensure delivery of the correct sequence ofsolutions.

In a preferred embodiment, the apparatus further comprises firstpressurization means for providing a positive pressure at the upstreamend of the filter relative to the downstream end thereof. The purpose ofthe pressurization means is to force or draw liquid in the first chamberthrough the filter so as to separate the liquid from cells or celldebris which are retained on the filter. This may be effected byapplying positive pressure upstream of the filter, for example by usingthe means for delivering the solutions to the first chamber to createpositive pressure in the first chamber. In a further arrangement, thepressure is lowered at the downstream end of the filter, for example byusing a vacuum line. In a still further arrangement, a syringe orsimilar means may reduce the pressure at the downstream end of thefilter and, at the same time, collect waste wash buffer.

Preferably, the means for retaining the solid phase matrix comprises abarrier to the solid phase matrix which is advantageously situatedbetween at least a part of the second chamber and the outlet. In thisarrangement, it is convenient for the solid phase matrix to be held atthe end of the second chamber downstream from the filtrate and permitdiscarded solutions to pass through the matrix and the barrier and toexit from the apparatus at the outlet. In an alternative arrangement,the outlet may be provided above the level of the barrier. In a furtherembodiment of the apparatus, the apparatus can be supplied with thesolid phase matrix already in position. The barrier is preferably afilter of the type described above. PTFE is a particularly usefulmaterial for this filter.

As an alternative, the barrier need not be a separate component but mayinstead arise simply from the geometry of the second chamber, forexample as a wall or compartment thereof.

In analogous fashion to the first pressurization means, the apparatusmay further comprise a second pressurization means for providing apositive pressure at the upstream end of the barrier relative to thedownstream end thereof. As discussed in relation to the firstpressurization means, either positive pressure at the upstream end ofthe barrier or negative pressure at the downstream end of the barriermay be applied. In one embodiment, a separate waste outlet is providedin addition to the outlet for the purified target compound. Negativepressure may be applied to the waste outlet, for example using a vacuumline for the purpose of removing unwanted liquids from the solid phasematrix.

Each of the outlets may be valve controlled and in the automatic versionof the apparatus, the valves would be linked to the control means.

Advantageously, the means for delivering solution and, when present, themeans for delivering suspensions and the pressurization means are drivenby a pneumatic fluid delivery system using continuous pressure,preferably continuous positive pressure.

Where phage-infected bacteria are used as the source material and lysedand unlysed bacterial cells are formed, a further embodiment of theapparatus may be required. In this embodiment the apparatus furthercomprises a pretreatment chamber upstream of the first chamber toreceive lysed and unlysed bacterial cells infected with phage and apretreatment filter, downstream of the pretreatment chamber and upstreamof the first chamber, for retaining unlysed bacterial cells andbacterial cell debris.

In a preferred embodiment, a single column arrangement is used in whichthe filter acts to partition the first chamber from the second chamber.In this type of arrangement the wall or walls defining the first chamberare continuous with those defining the second chamber. This has theadvantage that the arrangement can withstand relatively high operatingpressures such as those used in pneumatic delivery systems of the typedescribed herein. In a further embodiment, the first and second chambersare positioned side-by-side and the (vertical) filter again acts as apartition between the chambers. It is also possible to have the twochambers intercommunicating by means of tubing with in-line filtersseparate from the chambers.

In a particularly preferred embodiment, the apparatus is provided withfluid delivery means having an outlet which is positioned to communicatewith liquid when present in the second chamber. Typically, the liquidcollects at the bottom of the second chamber and the outlet ispositioned below the surface of the liquid. Whilst the fluid deliverymeans could act as the second pressurization means, it is preferablyseparate from the second pressurization means and capable of deliveringboth liquid and gas to the second chamber. The positioning of the outletenables gas to pass directly into the liquid in the second chamber so asto facilitate mixing of the components of the liquid. This isparticularly useful where a suspension or slurry of solid phase matrixis supplied to filtered lysate in the second chamber. Gas expelled fromthe outlet facilitates mixing of the solid phase matrix with thefiltered lysate to maximise contact of the target compound with thematrix.

The present invention will now be described further, by way of exampleonly, with reference to the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a simple manually-operated apparatus according to thepresent invention for plasmid DNA;

FIG. 2 shows a column version of the apparatus;

FIG. 3 shows an automated version of the apparatus;

FIG. 4 shows the results of agarose gel electrophoresis comparing DNAsamples prepared conventionally and samples in accordance with thepresent invention;

FIG. 5 shows a second column version of the apparatus; and

FIG. 6 shows a circuit diagram for operation of the apparatus.

In the simple embodiment of the invention shown in FIG. 1, cellreservoir 1 communicates with filtrate reservoir 7 through filter 2 andthree-way valve 3. Similarly, filtrate reservoir 7 communicates withoutlet 11 through filter 8 and three-way valve 9. Syringes 4 and 10 arealso connected respectively to three-way valves 3 and 9. Collection tube12 communicates with outlet 11 to collect the purified sample.

In the column embodiment of the invention shown in FIG. 2, the twochambers 21 and 27 are partitioned by filter 22. Further filter 28 actsas a barrier to prevent solid phase matrix in chamber 27 from passing tothe remainder of the second chamber 30 and to the outlet 31. In order todeliver solutions to the chambers 21 and 27, reagents from reservoirs(not shown) enter at inlets 33 to 38. The inlets are all controlled byvalves 23. Common ports 24 and 29 connect the reagent reservoirs withchambers 21 and 27 respectively. Vacuum lines can be applied to conduits25 and 26. Conduits 25 and 26 act additionally as waste outlets.Collection tube 32 communicates with outlet 31 to collect the purifiedsample.

A plurality of the columns can be assembled in the arrangement in FIG.3. Columns 41 and collection tubes 42 are shown. A reagent, fluiddelivery and control system 43 is present to control the process in eachcolumn.

The second column embodiment of the invention shown in FIG. 5 can beused in accordance with the circuit shown in FIG. 6.

Referring to FIG. 5, the two chambers 41 and 48 are partitioned byfilter 49. Further filter 48 acts as a barrier to prevent solid phasematrix in chamber 48 from passing out of the column. Inlet 42 isprovided in the first chamber of the column for the passage ofpressurised air and also to allow air to be vented from the column.Inlet 43 is provided for the addition of liquids to the column. Conduit47 consists of a conical portion connected to a tubular portion. Conduit47 collects liquid from filter 46 and transfers the liquid to acollection point typically at the base of the second chamber 48. Thebase of the second chamber includes a conical recess to maximise liquidcollection at the low volumes typically used in the apparatus. Line 44is provided at the top of the second chamber for passage of pressurizedair into the column and also to allow air to be vented from the column.A further inlet 45 is present for the addition of liquids or gas to thecolumn and consists of a tube with an outlet located near to the bottomof the column. Pinch valve 50 is situated downstream of filter 49 tocontrol flow from the second chamber and is monitored by means of liquidsensor 51. Indexing mechanism 54 controls the relative position of wastetube 53 or collection tube 52 according to whether liquid flowing fromthe column is intended for sampling or to be disposed of.

The liquid delivery system shown in FIG. 6 comprises a number ofreagents in reservoirs (R1 to R8) which are pressurized typically at 0.5bar fine pressure regulated air. The exact number of reagents used willdepend on the exact purification protocol to be adopted. The reagentsare divided into two blocks: reservoirs R1 to R4 contain reagents to bedelivered to inlet 43 of the column and are controlled by correspondingvalves 61 to 64; and reservoirs R5, R6 and R8 contain reagents to bedelivered to the second chamber of the column through inlet 45 undercontrol of valves 65 and 66. Reservoir R7 contains particulate matrixmaterial to be delivered by an independent line to the top of chamber 48through inlet 55 under control of corresponding valves 74 and 76.

Taking delivery of reagent from reservoir R4 as an example, this reagentis delivered by opening valve 64 for a selected time period (e.g. 250ms). Because the reservoir is under pressure and there is a drop inpressure on the open side of valve 64, reagent enters the system untilthe valve closes. There is now a volume of liquid in the line from valve64 to valve 68. The length and diameter of this line is chosen toaccommodate the maximum likely volume. The volume of reagent is thenmoved to the top of the first chamber by switching valve 68 on andswitching valve 67 on for sufficient time to allow the reagent to reachthe top of the chamber. The pressure of the air forcing the liquid downthe line can be either 0.5 bar or, if valve 77 is switched on, 1.7 bar.The air in front of the liquid to be moved is vented to atmosphere viavalves 69 and 70. All valve timings are controlled by a microprocessor(not shown).

Liquid flow to the column is controlled by flow constrictors C1 to C3(0.3 mm) or by using small diameter lines (0.5 mm or less--say valve 80to non-return valve NRV2). Non-return valves NRV1 to 4 are alsoincorporated in various parts of the circuit to prevent reverse flow.

By analogy with the delivery of reagent from reservoir R4, a similararrangement of valve control is used for delivery of reagent fromreservoirs R2, R3, R5 and R6. In order to wash the lines after additionof reagents from the reservoirs R2, R3 and R4 a volume of water isflushed through the lines from reservoir R1 in a similar manner as thedelivery of reagent but the water is diverted to waste at valve 68. Asimilar function is performed for washing the lines from reservoirs R5and R6 using water in R8 diverted to waste via valve 80.

Because of the particulate nature of the solid phase matrix reagent inreservoir R7, a separate delivery system is provided. This reagent isenclosed within a sealed reservoir and two lines go into the reagentwhile another is located at the top of the reservoir. In order to mixthe matrix prior to delivery and to ensure that it is in suspension,valve 76 is opened to allow air to escape via the vent. Because thereservoir is sealed to form a closed system, air is drawn in vianon-return valve NRV4 resulting in a bubbling action sufficient tosuspend matrix particles in the reagent. Valve 76 is then closed and anappropriate dose of the reagent from reservoir 7 is removed by openingpinch valve 74. The reagent is delivered to the top of the secondchamber of the column by opening valve 73 and venting via valve 71.

Where the transfer of liquid in the column from first chamber 41 tosecond chamber 48 is required, this is achieved by closing valves 68, 73and 72 and allowing air to enter the top of the first chamber via valve70. Any liquid in the first chamber is transferred to the second chamberwith air being vented via valve 71. Sensor 51 provided at the columnoutlet assesses whether liquid has passed out of the column via valve72. Indexing mechanism 54 is provided to move collection tube 52 intoposition to receive the product of the purification system.

EXAMPLE 1

In this Example the following materials were used.

    ______________________________________                                        Buffer (volumn/prep)                                                                        Composition                                                     ______________________________________                                        Resuspension Buffer                                                                         50 mM Tris-HCl pH 7.5                                             (300 ul) 10 mM Ethylenediamine tetra acetic                                    acid (EDTA)                                                                   100 g/ml RNase A                                                             Lysis Buffer 0.2 M NaOH, 1% Sodium dodecylsulphate                            (300 ul)                                                                      Neutralising Solution 2.55 M Potassium acetate                                (300 ul)                                                                      Matrix Wizard (formerly Magic) Minipreps                                      (500 ul) DNA purification resin in 7 M                                         Guanidine hydrochloride                                                       Wizard and Magic Minipreps are                                                registered trade marks of Promega                                             Corp.                                                                        Wash Buffer 200 mM Sodium chloride                                            (2000 ul) 20 mM Tris-HCl, pH 7.5                                               5 mM EDTA                                                                     50% ethanol                                                                  Elution Buffer 10 mM Tris-HCl, pH 7.5                                         (50 ul) 1 mM EDTA                                                           ______________________________________                                    

Referring to FIG. 1, a bacterial cell culture containing plasmid DNA wasadded to the cell reservoir 1 at a volume of 2 ml. The culture wasfiltered through filter 2 (8 micron cellulose acetate) via three-wayvalve 3 by means of a vacuum created by withdrawing the barrel ofsyringe 4 and collecting filtrate in syringe 4. The cells were left onthe surface of the filter 2. Syringe 4 was disconnected, the filtratediscarded and the syringe 4 was repositioned on three-way valve 3.Through the opening 5 successive additions of cell resuspension buffer,cell lysis buffer and cell neutralization solution were made. Betweeneach successive addition the cells were mixed by plunging syringe 4 toprovide plenty of turbulence. After the final addition, the entireliquid phase was filtered through the filter 2 and collected in syringe4. A solution of crude DNA was thus prepared.

Three-way valve 3 was then turned so that the entire contents of syringe4 could communicate with filtrate reservoir 7. The contents of syringe 4were then expelled into the filtrate reservoir 7 to which was added thematrix suspension. The crude DNA and matrix were mixed by moving syringe10 in and out at a distance equivalent to the void volume of the matrix.The entire contents of the filtrate reservoir 7 were then filteredthrough filter 8 (sintered PTFE) using syringe 10 to create a vacuum.Three-way valve 9 was then switched so that the filtrate could bediscarded. Wash buffer was added to the filtrate reservoir and voided aspreviously discussed. All residual wash buffer was removed by passingair through the filter 8 by means of plunger syringe 10. Finally,elution buffer was added to the filtrate reservoir 7, mixed by use ofthe syringe 10 and passed through filter 8. The purified DNA wasseparated from the resin in the elution buffer and collected incollection tube 12 by switching three-way valve 9.

The results of the present Example are shown in FIG. 4 as a comparisonwith results obtained using conventional centrifugation techniques. Forboth methods a starter culture of 5 ml plasmid-containing E. coli K12DH5 alpha was grown and 2 ml of the culture was used to prepare plasmidDNA. Two separate cultures were prepared by each method: one containingplasmid pBS SKII+; and one containing a clone of pBS SKII+containinginsert pFB41B2.

The results are shown in FIG. 4 in accordance with the following key.

    ______________________________________                                        Lane         Sample                                                           ______________________________________                                        M            MW marker lambda/Hind III                                          1 pBS SKII+/pFB41B2 (Example)                                                 2 pBS SKII+/pFB41B2 (conventional)                                            3 pBS SKII+ (Example)                                                         4 pBS SKII+ (conventional)                                                    M MW marker lambda/Hind III                                                 ______________________________________                                    

It will be appreciated from FIG. 4 that the yield from the presentinvention is comparable if not slightly greater than that obtained byconventional centrifugation Moreover, the purity of the DNA preparationsaccording to the present invention is at least comparable to thoseprepared by conventional methods. For example, DNA prepared according tothe present invention is ready for restriction enzyme analysis andmanipulation without the need to undergo further purification steps.

EXAMPLES II to IV

In these Examples the materials were used as shown in Table I.

                                      TABLE 1                                     __________________________________________________________________________    Composition of reagents for DNA purification                                    Examples II-IV                                                                   Example II III        IV                                                   Reagents Plasmid M13 Blood                                                  __________________________________________________________________________    1    de ionised water                                                                         de ionised water                                                                         de ionised water                                     2 50 mM Tris-HCl pH 7.5 Glacial acetic acid PBS                                10 mM EDTA                                                                    10 μg/ml RNAase A                                                         3 0.2 M NaOH 4 M NaClO.sub.4 20 mM Tris-HCl pH 8.0                             1% SDS  5 mM EDTA pH 8.0                                                     4 2.55 M Potassium acetate 70% Ethanol 50 mM KCl                                 10 mM Tris-HCl pH 8.3                                                         2.5 mM MgCl.sub.2                                                             0.45% NonIdet P-40                                                         5 200 mM NaCl 10 mM Tris-HCl pH 7.5 200 mM NaCl                                20 mM Tris-HCl pH 7.5 1 mM EDTA 20 mM Tris-HCl pH 7.5                         5 mM EDTA  5 mM EDTA                                                          70% Ethanol  70% Ethanol                                                     6 10 mM Tris-HCl pH 7.5  10 mM Tris-HCl pH 7.5                                 1 mM EDTA  1 mM EDTA                                                         7 Wizard DNA purification  Wizard DNA purification                             resin in 7 M Guanidine  resin in 7 M Guanidine                                HCl  HCl                                                                     Upper 1 μm cellulose acetate 1 μm cellulose acetate Leukosorb                                    type A or B                                          Filter                                                                        Lower 20 μm PTFE 1 μm borosilicate glass 1 μm borosilicate                                    glass                                                Filter                                                                      __________________________________________________________________________

Example II

Purification Protocol for Plasmid DNA

The column for purifying plasmid DNA has two filters: upper 1 um filter46 and lower 20 um filter 49. The material of the filter is chosen toresist the chemicals used during the process and to have appropriateflow characteristics to allow the process to occur as quickly aspossible. A freshly grown culture of E.coli containing a plasmid to bepurified is added to chamber 41, typically 1-5 ml. Addition could bemade by means of removing the top or by direct addition via a tube (notshown). Positive pressure is then applied to chamber 41 by means of port42. The bacteria are then filtered through filter 46 with the excessliquid passing to waste via conduit 47, filter 49 and valve 50. Theliquid waste is monitored by flow sensor 51. Once all liquid has passedthe flow sensor a dose of reagent 2 (TE/RNAase) is added to the top ofchamber 41 via inlet 3, typically 30 ul. To effect mixing, chamber 8 ispressurised in short bursts via line 44 and pressure in chamber 41 isvented via port 42. This is done to ensure that all the cells caught onthe filter are resuspended. A dose of reagent 3 (NaOH/SDS) is added tothe top of chamber 41 via inlet 43, typically 300 ul. This is then mixedas described for reagent 2. A dose of reagent 4 (KAcetate) is added tothe top of chamber 41 via inlet 43, typically 300 ul. This is then mixedas described for reagent 2. The resulting mixture is a cell free lysateof crude DNA with precipitated proteins. The cell free lysate of crudeDNA is then transferred from chamber 46 to chamber 48 by addition ofpositive pressure via port 42, venting chamber 48 via line 44 andclosing valve 50. A dose of reagent 7 (a DNA binding agent, e.g. silicaresins, diatomaceous earth, affinity matrix) is added to chamber 48 viatube 55, typically 500 ul. The DNA binding agent is then mixed with thecrude DNA by passing air into chamber 40 via line 45. The excess liquidis then removed from chamber 48 to waste. The DNA is retained on the DNAbinding agent which will not pass through filter 49. A dose of reagent 5(50% Ethanol/NaCl) is added to chamber 48 via line 45, typically 2000 uland passed through filter 49 and removed directly to waste by means ofpressurising chamber 48 with air from line 44. Air is continued to bepassed through filter 49 for a period that allows the filter to drysufficiently. The indexing mechanism 54 moves the collecting tube 52 sothat it is in line with the exit tube from chamber 48. The DNA is theneluted from chamber 48 by the addition of, typically, 50 ul of reagent6(TE) via lines 45 and expelled from chamber 48 by the addition ofpulses of pressure via line 44.

In between liquid additions the lines are rinsed out with water(reagent 1) and via valve 68 (FIG. 6).

EXAMPLES III

Purification Protocol for Ml3 DNA

Different reagents are used to purify DNA from Ml3 but the essentialelements of the apparatus are the same. The column for purifying singlestranded Ml3 DNA has two filters: upper 1 um filter 46; and lower 1 umfilter 49. The material of the filter is chosen to resist the chemicalsused during the process and to have appropriate flow/bindingcharacteristics to allow the process to occur as quickly as possible. Afreshly grown culture of E.coli containing Ml3 to be purified is addedto chamber 41, typically 1-5 ml. Positive pressure is then applied tochamber 41 by means of port 42. The bacteria are then filtered throughfilter 46 with the excess liquid containing Ml3 phage passing intochamber 48 via conduit 47. A dose of reagent 2 (Glacial acetic acid) isadded to the top of chamber 41 via inlet 43, typically 20 ul. The phageare then filtered through filter 49 with the excess liquid passing towaste via valve 50. The liquid waste is monitored by flow sensor 51.Once all liquid has passed the flow sensor, a dose of reagent 3 (4MNaClO₄) is added to the top of chamber 48 via line 45, typically 1000ul. The filter 49 is then washed with a dose of reagent 4 (70% EtOH),typically 1000 ul, and removed directly to waste by means ofpressurising chamber 48 with air from line 44. Air is continued to bepassed through filter 49 for a period that allows the filter to drysufficiently. The indexing mechanism 54 moves the collecting tube 52 sothat it is in line with the exit tube from chamber 48. The Ml3 DNA isthen eluted from chamber 48 by the addition of, typically, 50 ul ofreagent 5 (TE) via line 45 and expelled from chamber 48 by the additionof pulses of pressure via line 44.

EXAMPLES IV

Purification Protocol for Genomic DNA from Blood

The column for the purification of genomic DNA from blood has twofilters: upper filter 46 is a Leukosorb type A or B (Pall Biosurportdivision); and lower filter 49 is a 20 um filter. The material of theupper filter was chosen so that it would retain nucleated cells whileallowing denucleated red blood cells through. Freshly collected bloodwith EDTA as anti-coagulant was added to chamber 41 typically 1-10 ml.This could be done by means of removing the top or by direct additionvia a tube (not shown).

Positive pressure is then applied to chamber 41 by means of inlet 42.The red blood cells are then filtered through filter 46 with the excessliquid passing to waste via conduit 47, filter 49 and valve 50. Theliquid waste is monitored by flow sensor 51. Once all liquid has passedthe flow sensor a dose of reagent 2 (PBS) is added to the top of chamber41 via line 43, typically 2000 ul. To effect mixing chamber 48 ispressurised in short bursts via line 44 and chamber 41 is vented viainlet 42. This is done to ensure that cells caught on the filter areresuspended. A dose of reagent 3 (20TE) is added to the top of chamber41 via line 43, typically 2000 ul, to lyse any remaining red bloodcells. This is then mixed as described for reagent 2. The white bloodcells are then lysed with a dose of reagent 4 (lysis buffer) which isadded to the top of chamber 41 via line 43, typically 500 ul. This isthen mixed as described for reagent 1. The resulting mixture is a cellfree lysate of crude DNA. This is then transferred from chamber 41 tochamber 48 by addition of positive pressure via port 42, venting chamber48 via line 44 and closing valve 50. A dose of reagent 7 (a DNA bindingagent, e.g. silica resins, diatomaceous earth, affinity matrix) is addedto chamber 48 via tube 55, typically 500 ul. The DNA binding agent isthen mixed with the crude DNA by passing air into chamber 48 via line45. The excess liquid is then removed from chamber 48 to waste. The DNAis retained on the DNA binding agent which will not pass through filter49. A dose of reagent 5 (50% Ethanol/NaCl) is added to chamber 48 vialine 45, typically 2000 ul and passed through filter 49 and removeddireclty to waste by means of pressurising chamber 48 with air from line44. Air is continued to be passed through filter 49 for a period thatallows the filter to dry sufficiently. The indexing mechanism moves thecollecting tube 52 so that it is in line with the exit tube from chamber48. The DNA is then eluted from chamber 48 by the addition of pulses ofpressure via line 44. In between liquid additions the lines are rinsedout with water (reagent 1) via valve 68 (FIG. 6).

    ______________________________________                                        Key to FIG. 6                                                                   Symbols                      Numbers                                        ______________________________________                                                    2 way valve normally closed                                                                      61, 62, 63,                                        64, 65, 66,                                                                   75                                                                            2 way valve normally open 69, 73                                              3 way valve for liquid 68, 80                                                 3 way valve for air 67, 70, 71,                                               76, 77, 78,                                                                   79                                                                            3 way pinch valve for air 74                                                  2 way pinch valve normally open 79                                            Liquid column detector LCD1                                                   Non return valve NRV1,                                                        NRV2,                                                                         NRV3,                                                                         NRV4                                                                          Reagent bottles R1, R2, R3,                                                   R4, R5, R6,                                                                   R8                                                                            Reagent bottle R7                                                             Flow constrictor 0.3 mm i.d. C1, C2, C3                                       Compressed air cylinder actuator                                              Air filter 0.2 μm                                                          Air filter 5 μm                                                            Pressure regulator with gauge                                                 Variable flow control                                                         Compressed air source                                                         Air vent                                                                      Line to waste                                                                 Tube, inside diameter 3 mm                                                    Tube, inside diameter 0.8 mm                                                  Tube, inside diameter 0.5 mm                                              ______________________________________                                    

What is claimed is:
 1. A method for continuous purification of nucleicacid from cells, which method comprises the following steps:(1) applyinga crude cell suspension from a first chamber, delimited by a wall, to afilter to separate the cells from medium and resuspending the cells inthe first chamber to form a cell suspension; (2) lysing the cellsuspension in the first chamber to form a cell lysate containing nucleicacid; (3) applying the cell lysate to the filter to remove unwantedcells and cell debris; (4) contacting the filtered lysate with a solidphase matrix retained in or downstream of a second chamber, delimited bya wall, under conditions to bind the nucleic acid to the matrix; (5)separating the resultant filtered lysate from the matrix; and (6)applying liquid to the matrix through solution delivery meanscommunicating with the second chamber so as to elute the nucleic acidfrom the matrix through an outlet which is provided downstream of thematrix; wherein the walls of the first and second chambers arecontinuous with one another; wherein the step of resuspending the cellsin the first chamber comprises adding resuspension buffer to the cellsand producing a pressure difference across the filter to resuspend thecells retained by the filter so as to form the cell suspension; andwherein the method does not include a centrifugation step.
 2. The methodaccording to claim 1, wherein the nucleic acid is DNA.
 3. The methodaccording to claim 2, wherein the DNA is plasmid DNA.
 4. The methodaccording to claim 2, wherein the cells are blood cells.
 5. The methodaccording to claim 4, wherein the crude cell suspension comprises blood.6. The method according to claim 1, wherein the solid phase matrix isretained by a second filter.
 7. The method according to claim 1, whereinthe matrix and bound nucleic acid are dried by passage of air prior toelution.
 8. The method according to claim 1, which further comprises thestep of washing the matrix after binding the nucleic acid and beforestep (5) to remove contaminants before eluting the nucleic acid from thematrix.
 9. The method according to claim 1, wherein each filter has apore size in the range 0.2 to 50 microns.
 10. The method according toclaim 1, wherein the step of contacting the filtered lysate with thesolid phase matrix comprises adding a suspension of the solid phasematrix to the filtered lysate.
 11. The method according to claim 1,wherein the solid phase matrix comprises an ion exchange material. 12.The method according to claim 11, wherein the ion exchange material isan anion exchange material.
 13. The method according to claim 1, whereinthe nucleic acid is kept in solution when not bound to the solid phasematrix.
 14. An apparatus for the continuous purification of a targetcompound from a crude cell suspension comprising:a first chamber forreceiving the crude cell suspension, for receiving a cell suspension ofre-suspended cells, and for receiving cell lysate formed from lysed cellsuspension; a filter downstream of the first chamber for separatingcells from medium and retaining unlysed cells and cell debris; a secondchamber for receiving filtered lysate downstream of the filter; meansfor retaining a solid phase matrix to bind the target compound providedin or downstream of the second chamber; solution delivery meanscommunicating with the second chamber for eluting the target compoundfrom the matrix; an outlet for delivering the purified target compoundprovided downstream of the means for retaining the solid phase matrix;and fluid delivery means having an outlet which is positioned tocommunicate with liquid when present in the second chamber; wherein thewalls of the first and second chambers are continuous with one another;and wherein means are provided for producing a pressure differenceacross the filter to re-suspend cells retained by the filter and formthe cell suspension.
 15. The apparatus according to claim 14, whereinthe first chamber communicates with further means for deliveringsolutions thereto.
 16. The apparatus according to claim 15, wherein themeans for delivering solutions is driven by a pneumatic fluid deliverysystem using continuous pressure.
 17. The apparatus according to claim14, further comprising first pressurization means for providing apositive pressure at the upstream end of the filter relative to thedownstream end thereof.
 18. The apparatus according to claim 17, whereinthe first pressurization means is driven by a pneumatic fluid deliverysystem using continuous pressure.
 19. The apparatus according to claimsclaim 14, wherein the means for retaining the solid phase matrixcomprises a barrier to the solid phase matrix, which barrier is situatedbetween at least a part of the second chamber and the outlet.
 20. Theapparatus according to claim 19, further comprising secondpressurization means for providing a positive pressure at the upstreamend of the barrier relative to the downstream end thereof.
 21. Theapparatus according to claim 14, wherein the second chamber communicateswith means for delivering thereto a suspension the solid phase matrix.22. The apparatus according to claim 21, wherein the means fordelivering suspensions is driven by a pneumatic fluid delivery systemusing continuous pressure.
 23. The apparatus according to claim 14,wherein a conduit is provided to transmit the filtrate from the filterto a collection point in the second chamber.
 24. The apparatus accordingto claim 14, wherein each means for delivering solutions and, whenpresent, the means for delivering suspensions and the pressurizationmeans are driven by a pneumatic fluid delivery system using continuouspressure.
 25. The apparatus according to claim 14, which furthercomprises control means for coordinating delivery of solutions,suspensions and pressure.
 26. The apparatus according to claim 14,wherein the means for producing a pressure difference across the filtercomprise a fluid line to pressurize the second chamber.
 27. Theapparatus according to claim 14, wherein the means for retaining a solidphase matrix comprises a second filter.
 28. The apparatus according to14, wherein means are provided for drying the matrix by air.
 29. Theapparatus according to claim 14, which further comprises a three-wayvalve downstream of the second chamber and upstream of the outlet forcontrolling elution from the apparatus.